Summary Synovial joints are critical for skeletal form, flexibility, and function, and are susceptible to debilitating diseases such as osteoarthritis and injury. While much is known about the composition and mechanics of functioning joints, relatively little is known about how joint-forming cells are specified in a particular location. We use the zebrafish regenerating fin to address fundamental questions of skeletal development, including the specification and commitment of the cells producing fin ray joints. The fin skeleton is made of bony fin rays, and each fin ray is made of bony segments separated by joints. The osteoblasts and joint-forming cells that build the fin skeleton are derived from a common skeletal precursor cell located in the lateral fin ray mesenchyme. Recent studies from the Iovine lab provide strong evidence that the gap junction protein Connexin43 (Cx43) influences the location of fin ray joints, and further, that Cx43 influences the expression of the evx1 transcription factor required for specification of joint-forming cells. Our primary objective for this proposal is to provide mechanistic insight into how Cx43 influences evx1 expression. Progress from the current funding period demonstrates that cx43 mRNA levels are transiently reduced coincident with the initiation of each new fin ray joint, and that manipulating Cx43 influences both evx1 expression and joint formation. Moreover, we find that Cx43 activity in the medial fibroblasts is responsible for influencing joint formation in the lateral mesenchyme. Thus, the underlying hypothesis of this proposal is that Cx43 function in the medial fibroblasts determines when evx1 expression is up-regulated in the lateral skeletal precursor cells, and therefore determines when and where a joint is produced. The Aims of this proposal are designed to test this hypothesis. In Aim 1 we will identify the non-autonomous function of Cx43, including how Cx43 function is regulated in the medial fibroblasts. In Aim 2 we will provide insights into the molecular pathway(s) acting between Cx43 activity and evx1 transcription. Completion of this proposal will provide novel insights into the molecular mechanisms underlying where and how joint-forming cells are specified, and will therefore enhance our understanding of fundamental questions regarding skeletal form and function.